CD4 is a surface molecule that serves as the receptor for human immunodeficiency virus (HIV), the causative agent of AIDS. We are studying two areas related to CD4 and HIV: 1) Mechanisms of membrane fusion mediated by the CD4/HIV env interaction. One focus of this work is to identify accessory components of CD4+ target cells which determine the specificity of HIV fusion. Two types of specificities are under study: the selective tropism of different HIV-1 isolates for T cell lines vs. primary macrophages, and the ability of human CD4 to mediate fusion when expressed on a human cell but not on an animal cell. To study these problems, we have developed a novel vaccinia-based assay in which fusion between CD4+ and env+ cells leads to lacZ gene activation by phage T7 RNA polymerase and consequent accumulation of beta-galactosidase in the cytoplasm of the fused cells. The rapidity, sensitivity, and flexibility of this assay make it highly suitable for mechanistic studies and for screening fusion-blocking antibodies and drugs. Using this assay, we have obtained the first direct evidence that the replication tropism of different HIV-1 isolates for T cell lines vs. primary macrophages is determined primarily by the specificity of env-mediated fusion with the target cell. Regarding the species specificity, we have demonstrated functional transfer the human cell-specific accessory component(s) to animal cells using PEG-mediated membrane vesicle fusion. The identities of the cellular components associated with specificity of env/CD4-mediated fusion are a major focus for present work. We also observed that co-expression of the protease furin greatly stimulates env/CD4 fusion in the vaccinia system, presumably by enhancing processing of the gp160 precursor. This finding greatly enhances the cell fusion assay system and should help circumvent problems which have compromised a second focus of this work, namely analysis of structural features of env and CD4 in fusion. By co-expressing furin, we expect to be able to produce large quantities of correctly processed env glycoprotein (membrane-associated and soluble forms) for structure/function studies. 2) CD4-based therapeutics for treatment of HIV infection. We have designed a genetically engineered hybrid toxin (CD4-PE40) which selectively kills HIV-infected cells and is active against both laboratory-adapted and primary HIV-1 isolates. We are currently testing whether the dose-limiting hepatotoxicity observed in initial Phase I clinical trials is associated with gp120 binding to CD4-PE40 and consequent enhanced uptake into liver cells. Studies are also in progress to test the ability of CD4-PE40 (alone or in combination with other antiretroviral agents) to eliminate HIV ex vivo from infected PBLs and bone marrow cells obtained from HIV-positive individuals. Such methods may prove useful in genetic therapy and bone marrow transplantation approaches to combat AIDS.